Materials and methods in quantifying cell mechanobiology

时空机械基质和力显微镜的进步量化细胞收缩性和粘弹性。在过去的几十年里，生物系统的力和机械特性被认为是所有生命尺度的基本组成部分。特别是，弹性细胞培养基质的出现催化了细胞生物学的复兴，解锁了生物科学中以前未被认识的维度。理解驱动生物过程的物理机制是21世纪的一个关键挑战，并将通过力和力学的全新视角来解决生物系统，然而，量化这些相互作用的材料和方法是必不可少的。大多数真核细胞的活性特性的核心是它们是可收缩的，不断地对邻近的细胞和它们的底物施加动力。通过在可变形基质上培养细胞，研究人员利用牵引力显微镜（TFM）来表征细胞的刚度依赖的收缩性，以及这种收缩性对从分化、增殖到癌症转移的各种生物过程的影响。然而，衬底稳定性、孔隙度、通量和技术复杂性等瓶颈阻碍了这些基本指标在更广泛的生命科学应用中的采用。我的实验室已经生产出用于在不同生理和病理环境下进行高通量收缩力测量的机械可调硅基板，NSERC DG的支持对这些进展至关重要。该DG提案的重点是通过开发(1)量化细胞力学的新材料和新方法来扩展这一前沿领域，目标是将这些应用于(2)量化细胞粘弹性。为此，我们将创建硅基电池衬底，a)在空间中具有弹性图案；B)时间上的弹性转换；C)粘附模式直接单个和集体细胞结构维度；d)局部收缩拉伸细胞并探测其机械反应的水凝胶基质。我的实验室将以我们在TFM方面的专业知识为基础，简化和提高实时细胞收缩性和机械性能的量化。我们将选择性地修饰关键的细胞内和细胞间蛋白，并量化它们对收缩力和细胞粘弹性的贡献。这个DG项目是建立在我的实验室在硅胶衬底力学和细胞力测量方面的专业知识的坚实基础上的，并得到了广泛的显微镜和机械表征仪器的良好支持。我的DG项目将推动基于材料的细胞收缩性和粘弹性的量化。由于这里提出的创新的广泛和基础实用性，我预计在生物物理学、定量生物学、材料科学和实验医学方面会产生深远的影响。

Spatiotemporal mechanical substrates and force microscopy advances to quantify cell contractility and viscoelasticity. Over the past decades, the forces and mechanical properties of biological systems have been recognized as an essential component across all scales of life. In particular, advents in elastic cell culture substrates have catalyzed a renaissance of cell biology, unlocking a previously unrecognized dimension in biological sciences. Understanding the physical mechanisms driving biological processes is a key challenge of the 21st century, and will resolve biological systems through an entirely new perspective of forces and mechanics, however, materials and methods to quantify these interactions are essential. Central to the active properties of most eukaryotic cells is that they are contractile, continuously exerting dynamic forces that pull on neighboring cells and their substrate. By culturing cells on deformable substrates, researchers have employed Traction Force Microscopy (TFM) to characterize the stiffness-dependent contractility of cells and the influence this contractility has on diverse biological processes from differentiation and proliferation to cancer metastasis. Bottlenecks of substrate stability, porosity, throughput, and technical complexity nevertheless have hampered adoption of these essential metrics in broader life-science applications. My lab has produced mechanically tunable silicone substrates for high-throughput contractile force measurements in diverse physiological and pathological contexts, and NSERC DG support has been critical in these advances. This DG proposal is focused on extending this frontier by developing (1) new materials and methodologies to quantify cell mechanics, with the goal of applying these to (2) quantify cell viscoelasticity. To do so, we will create silicone-based cell substrates that are a) elastically patterned in space; b) elastically switchable in time; c) adhesively patterned to direct single and collective cell structure dimensionality; and d) hydrogel substrates that locally contract to stretch cells and probe their mechanical response. My lab will build on our expertise with TFM to both simplify and enhance quantification of real-time cell contractility and mechanical properties. We will selectively modify key intracellular and intercellular proteins and quantify their contributions to contractile forces and cellular viscoelasticity. This DG program is built on a firm foundation of my lab's expertise in silicone substrate mechanics and cell force measurements, and well-supported by extensive microscopy and mechanical-characterization instrumentation. My DG program will drive a materials-based quantification of cell contractility and viscoelasticity. Due to the broad and fundamental utility of the innovations proposed here, I anticipate far-reaching impact in biological physics, quantitative biology, materials science, and experimental medicine.